Malaria-specific DNA sequences, expression products thereof, and the use thereof

ABSTRACT

The invention relates to malaria-specific nucleic acid sequences, to the expression products thereof, and to the use thereof. A combination of three of the expression proteins whose nucleic acid sequences were isolated by screening a lambda gt11 gene bank with a monospecific antiserum against the protective 41 kD antigen band from P. falciparum protects Aotus monkeys from a P. falciparum infection in model experiments.

This application is a continuation of U.S. patent application Ser. No.07/989,641 filed Dec. 11, 1992, now abandoned, which is a divisional ofU.S. patent application Ser. No. 07/723,668, filed Jun. 27, 1991, nowU.S. Pat. No. 5,194,587, which is a continuation of U.S. patentapplication Ser. No. 07/290,942, filed Dec. 28, 1988, now abandoned.

An important step towards the development of a vaccine against malariais the identification of protective antigens. In general, the antigenscategorized as protective are those which have provided protection fromP. falciparum infection administered intravenously in in vivoexperiments in an animal model such as, for example, in Saimiri or Aotusmonkeys. Only unsatisfactory protection experiments have hitherto beendescribed in humans, but several isolated P. falciparum proteins haveshown a complete or partial protective effect in an animal model. Thisapplies both to protein fractions of 75 kD and 100 kD purified by gelelectrophoresis and to the protein bands of molecular weights 200 kD,140 kD and 41 kD purified by gel electrophoresis (L. H. Perrin et al.(1984), Clin. exp. Immunol. 56, 67-72; L. H. Perrin et al. (1985), J.Clin. Invest. 75, 1718-1721; W. A. Siddiqui et al. (1987), Proc. Natl.Acad. Sci., USA 84, 3014-3018). Of the proteins which are specific formerozoites and have been prepared to date by biotechnological methods, apartial protective effect in immunization experiments with Saimiri orAotus monkeys has been shown by a recombinant expression protein of the5' repeat region of the so-called RESA 155 kD merozoite protein as wellas by a synthetic oligopeptide of the 200 kD merozoite surface precursorprotein and by a combination of synthetic oligopeptides of proteins ofmolecular weights 35 kD, 55 kD and 200 kD. Recombinant proteins,prepared by genetic manipulation, of the above antigens, which display aprotective effect in in vivo experiments with monkeys, are potentialcandidates for a malaria vaccine.

The aim of the investigations was to isolate coding sequences for theprotective 41 kD antigen band described by L. Perrin (1985 loc. cit.),to bring about the expression of the sequences, and to test theexpression products for their protective effect in the monkey model. Aspecific antiserum against the 41 kD antigen band was used to isolatefrom a genomic expression bank fifteen clones and to elucidate thestructure of their insertions. The sequences of the clones 41-1 to 41-10and 41-12 to 41-15, as well as 41-17, are depicted in Tab. 1-15. Twoclones (41-2 and 41-7) with very intense immunological reactions wereused to isolate mono-specific antibodies from the serum used for thescreening. These antibodies react specifically in the Western blot witha merozoite antigen of 41 kD.

In the Southern blot, a 3.0 kb EcoRI fragment and a 2.0 kb Sau3Afragment hybridized with the insert DNA of the clone 41-2. Both DNAfragments were isolated and sequenced.

The Sau3A fragment contains the complete coding region of the 41-2 gene.This region contains no introns and codes for 184 amino acids with amolecular weight of 21512 D. The 41-2 protein possesses a signalsequence and two further hydrophobic sections. No repetitive sequenceportions are present. Western blot analysis of schizont proteins withrabbit antisera prepared against an expression product which contains70% of the coding region produced a band of 29 kD. Furthermore, an mRNAof 1.6 kb was detected by Northern blot analysis.

On the other hand, the insert DNA of the clone 41-7 codes for the 41 kDprotein. Rabbit antisera prepared against a fusion protein of 41-7unambiguously recognize a 41 kD band in the Western blot. It waspossible, by screening a genomic lambda gt11 EcoRI* gene bank with theinsert DNA of the clone 41-7, to identify a clone which contains amalaria-specific insert of 2.3 kb. This was isolated and sequenced. Itcontains the complete coding region for a 41 kD protein. The gene doesnot encode a signal sequence and contains neither introns nor repetitivesections. The derived amino acid sequence of the 41 kD protein from P.falciparum is highly homologous (>60%) with aldolases from muscle andliver of mammals and with the aldolase from Trypanosoma brucei. Incontrast to the mammalian genome, only one aldolase gene per genome wasfound by Southern blot analyses for P. falciparum.

The clones 41-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 and 17 weredetected on the basis of their cross-reactivities with the antiserumagainst the 41 kD protein band. They are suitable for the preparation ofa vaccine. Expression of the insert DNAs of the clones 41-1 to 41-5 and41-7, 41-10 and 41-14 was brought about in the vector pEX-31, and theresulting fusion proteins were purified. A combination of animmunologically effective amount of three expression products (41-1,41-2 and 41-3) protects Aotus monkeys from P. falciparum infection.

Consequently, the invention relates to

a) the purified and isolated DNA sequences of the clones 41-1 to 41-10,41-12 to 41-15 and 41-17, as well as 41-gen and 41-7gen, including theirtranscription products,

b) DNA structures and vectors containing these sequences in whole or inpart,

c) pro- or eukaryotic cells transformed with such DNA,

d) the polypeptides, or parts thereof, expressed by these cells byreason of the transformation,

e) the amino acid sequences thereof,

f) antibodies against the polypeptides under (d), including the usethereof for passive immunization, for diagnosis and for purifying saidpolypeptides,

g) vaccines against malaria which contain the amino acid sequences from(e) alone or in combination,

h) a process for the preparation, by genetic manipulation, of thepolypeptides, or parts thereof, detailed under (d),

i) and the use of the said amino acid sequences for diagnosis.

Further embodiments of the invention are detailed in the examples andtables which follow and in the patent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the course of parasitemia in three Aotus monkeys afterimmunization with a combination vaccine composed of fusion proteins ofthe clones 41-1, 41-2 and 41-3. The Y axis represents percentage ofparasitemia; the abscissa represents time after infection.

FIG. 1B depicts the course of parasitemia in three non-immunized Aotusmonkeys.

EXAMPLES Example 1

Screening of a lambda gt11 expression bank using the mono-specificanti-41 kD serum

10⁶ PFU (plague-forming units) of a genomic lambda gt11 expression(prepared from DNA of the P. falciparum strain T996) were screened withan antiserum against the 41 kD antigen band (L. H. Perrin et al. (1985)loc. cit.) from the P. falciparum strain SGE2 by known methods (L. S.Ozaki (1986), J. Immun. Method. 89, 213-219; Promega Biotec (1986),Proto Blot Immunoscreening System, Technical Manual). The detectionsystem used for this was an anti-rabbit IgG/alkaline phosphataseconjugate (Promega, Order No. P 3731).

The screening of the genomic lambda gt11 gene bank with the antiserumagainst the 41 kD protein band made it possible to identify two veryintensely reacting clones (41-2 and 41-7) and thirteen other cloneswhich reacted more weakly (41-1, 41-3, 41-4, 41-5, 41-6, 41-8, 41-9,41-10, 41-12, 41-13, 41-14, 41-15 and 41-17) and about 40 other veryweakly reacting clones. The insert DNAs of the fifteen clones, whichamount to 140 bp to 650 bp, were cut out with EcoRI and cloned into theEcoRI site of the vector pUC8 for further characterization.

Example 2

Sequencing of the insert fragments of clones 41-1 to 41-10, 41-12 to41-15 and 41-17

The insert DNAs were sequenced by the dideoxy method using a primer anda reverse primer directly from the pUC8 plasmids (E. Y. Chen and P. H.Seeburg (1985), DNA 4, 165-170).

Tables 1-15 show the malaria-specific DNA sequences, and the amino acidsequences derived therefrom, of the clones 41-1, 41-2, 41-3, 41-4, 41-5,41-6, 41-7, 41-8, 41-9, 41-10, 41-12, 41-13, 41-14, 41-15 and 41-17 inthe only possible open reading frames. There are no overlaps orhomologies whatever in these 15 sequence sections. The UWGCG (Universityof Wisconsin, Genetic Computer Group) program was used to examine these15 sequences for homologous sequence sections within the EMBL data bank.None of these 15 partial sequences or relatively large homologoussections have hitherto been described. There is merely a 74% homology ofnucleotides 1 to 134 in the sequence of the clone 41-10 with a partialsequence from nucleotide 2144 to 2274 in the 140 kD protein gene, asproposed in the Application DE-P 3,741,057. The sequence of the clone41-10 is also the only one which contains repetitive sequence sectionstypical of P. falciparum proteins. The amino acid sequence of this cloneincludes three tetrapeptides of the sequence Pro-Ser-Glu-Ser, with thesecond serine residue in the second repeat being replaced by anasparagine residue, caused by a G-A transition. In addition, thesequence of the clone 41-7 from nucleotide 50 to 163 is 56% homologouswith an aldolase mRNA from nucleotide 218 to 331 of the rat (T. Mucai etal. (1986), J. Biol. Chem. 261, 3347-3354).

Example 3

Detection of the 41 kD antigen using specific antibodies against theexpression clones 41-2 and 41-7

The method of L. S. Ozaki (1986, loc. cit) was used to isolate from theantiserum against the 41 kD protein band antibodies which are directedspecifically against the products of the expression clones 41-1, 41-2,41-3, 41-7, 41-8, and lambda gt11 (control). To obtain schizonts, P.falciparum was cultivated in human erythrocytes (W. Trager and J. B.Jensen (1976), Science 193, 673-675) and synchronized by treatment withsorbitol (C. Lambros and J. P. Vanderberg (1979), J. Parasitol. 65,418-420). The schizonts were enriched to about 90% by flotation inGelafundin® (Braun Melsungen) (in analogy to G. Pasvol et al. (1978),Ann. Trop. Med. Parasitol. 72, 87-88). The schizonts were removed bycentrifugation, washed, heated at 100° C. in SDS sample buffer for 5min, treated with ultrasound, and frozen in aliquots.

Aliquots of the schizont solution were used for Western blot analysis ofthe abovementioned specific antibodies (D. A. Johnson et al. (1984) GeneAnal. Tech. 1, 3-8). In this the antibodies which were isolated usingthe expression clones 41-2 and 41-7 reacted very intensely with a 41 kDantigen band from schizonts.

Example 4

Cloning of a DNA fragment which contains the genetic information of theclone 41-2

15 μg of genomic DNA of the P. falciparum strain FCBR, which had beenobtained by lysis of schizont cultures followed by ethidium bromide/CsClcentrifugation (P. Oquendo et al. (1986) Molecular and BiochemicalParasitology 18, 89-101), were digested with the restriction enzymeEcoRI, blotted onto Gene Screen membranes (Dupont) in accordance withthe manufacturer's instructions, and then hybridized withnick-translated insert DNA of the clone 41-2 with a specific activity of10⁷ to 10⁸ dpm/μg. After the filter had been washed in 0.3×SSC(1×SSC=0.15M NaCl, 0.015M Na citrate) and 1% SDS (sodium dodecylsulfate) at 65° C. for 1 h, the filters were autoradiographed. ThisSouthern blot experiment was used to identify a genomic EcoRI fragmentwhich is about 3 kb in size and hybridizes with the insert DNA of theclone 41-2. 60 μg of P. falciparum DNA of the strain FCBR which had beencut with the restriction enzyme EcoRI were fractionated in a preparativegel, and the region from 2.8 kb to 3.2 kb was cut out and electroeluted(B. Perbal (1984), A Practical Guide to Molecular Cloning). This DNA wascloned by the method of T. V. Huynh et al. (in DNA cloning Vol. I, ed D.M. Glover (1985), 49-88) into the vector lambda gt10. 10⁵ PFU of theresulting gene bank were screened with nick-translated insert DNA of theclone 41-2 by known methods (T. Maniatis et al. (1982), MolecularCloning, A Laboratory Manual). This resulted in several phage cloneswhich hybridized with the insert DNA of the clone 41-2. The phage DNA ofone of these clones was isolated (R. W. Davis et al. (1980), A Manualfor Genetic Engineering, Advanced Bacterial Genetics) and digested withthe restriction enzyme EcoRI; a DNA fragment 3.0 kb in size was purifiedby gel electrophoresis and subcloned into the EcoRI restriction site ofthe vector pUC18 (plasmid pUC 41-2gen). In the subsequent Southern blotanalysis (T. Maniatis et al., loc. cit.), this 3.0 kb EcoRI DNA fragmentof pUC 41-2gen hybridized with the insert DNA of the clone 41-2.

Example 5

Sequence analysis of the clone pUC 41-2gen

The plasmid DNA pUC 41-2gen was sequenced starting from the EcoRI edgesites and using a primer and reverse primer (E. Y. Chen and P. H.Seeburg (1985) loc. cit.). From this it was possible to determine about250 bases from each of the ends of the 3.0 kb EcoRI fragment. Thesequence of one of these ends is identical to the insert DNA of theclone 41-2. To construct a restriction map, 0.5 μg samples of theisolated 3.0 kb EcoRI DNA fragment were incubated with variousrestriction enzymes, fractionated by gel electrophoresis, blotted ontonitrocellulose and hybridized with nick-translated insert DNA of theclone 41-2 by known methods (T. Maniatis, loc. cit.). The size of therestriction fragments to be hybridized made it possible to deduce thedistance of various restriction cleavage sites from the EcoRI cleavagesite, which is common to the two clones 41-2 and pUC 41-2gen. Based onthe restriction map constructed in this way, restriction fragments ofthe clone pUC 41-2gen were isolated and subcloned into the phage vectorsM13mp18 and M13mp19 for sequencing (F. Sanger et al. (1977), Proc. Natl.Acad. Sci. USA, 74,5463-5467). From this, the sequence was determinedfrom the EcoRI restriction site, which belongs to the gene, in thedirection towards the 5' end of the gene up to a DraI restrictioncleavage site. Table 16 shows the DNA sequence and the derived aminoacid sequence of this DraI-EcoRI DNA fragment comprising 1230 bp of theclone pUC 41-2gen. The sequence from position 1036 to 1228 is identicalto the insert DNA of 41-2. Since the sequence of the clone 41-2 and thegenomic sequence of the clone pUC 41-2gen derive from different P.falciparum strains (strain T996 from Thailand and strain FCBR fromColumbia), it appears that at least this gene section is highlyconserved. The open reading frame of this sequence begins in position784 with an ATG start codon and terminates with a TTC codon whichbelongs to the EcoRI restriction cleavage site within the gene. Thispart codes for the 149 N-terminal amino acids of the protein. Thepartial sequence of this gene has no repetitive sequence segments. Thederived amino acid sequence begins with a sequence section of 18 aminoacids, of which 4 are acidic and 5 are basic. This sequence section isfollowed by a hydrophobic part which is composed of 11 residues and isflanked on both sides by acidic amino acid residues. This hydrophobicregion might function as a signal sequence. The region with basic andacidic amino acids in front of this putative signal sequence isrelatively long; however, regions of similar length have also beendescribed for other P. falciparum proteins (T. Triglia et al. (1987),the EMBO Journal 6, 1413-1419). The derived amino acid sequence wasexamined, using the UWGCG program, for hydrophilic regions and surfaceregions as well as for potential immunogenic epitope regions. Thisrevealed three hydrophilic regions of the protein which are encoded bythe nucleotide sequences of positions 890 to 907, 1079 to 1093 and 1151to 1168.

The 5' non-coding region of the gene is extremely AT-rich (ATcontent=88.8%), as has also been described for other P. falciparum genes(J. L. Weber (1987), Gene 52, 103-109), and has in each case more than15 stop codons in each of the three reading frames. Furthermore, apossible CAAT box is present in position 274, and a possible TATA box islocated 64 nucleotides downstream of this. These structures mightspecify a possible promoter region for this gene.

Example 6

Isolation of the complete gene for 41-2

The sequence analysis of the clone pUC 41-2gen revealed a Sau3A cleavagesite 947 bp away from the EcoRI cleavage site. Southern blot analysis ofthe genome identified a Sau3A fragment which is 2.0 kb in size andhybridizes with the insert DNA of the clone 41-2 (cf. Example 4). Hencethis Sau3A fragment ought to have about 1050 bp of the geneticinformation of the 41-2 gene in the 3' direction from the EcoRI site. 60μg of DNA from the strain FCBR which has been cut with the restrictionenzyme Sau3A were fractionated in a preparative gel, and DNA fragmentsof 1.8 kb to 2.2 kb were isolated. This DNA was cloned into the XhoIsite of the vector lambda ZAP as stated by the manufacturer(Stratagene). 10⁵ PFU of this gene bank were screened withnick-translated insert DNA of the clone 41-2, and about 40 phage cloneswere identified (cf. Example 4). By automatic excision using theStratagene method, a bluescript vector (pSK⁻ 41-2gen) was isolated fromone of these phage clones. Restriction of this plasmid DNA with KpnI andEcoRI resulted in the isolation of two DNA fragments of 950 bp and 1050bp, of which the 950 bp fragment hybridized in the Southern blot withthe insert DNA of the clone 41-2. The plasmid pSK⁻ 41-2 DNA wassequenced using a primer and reverse primer (E. Y. Chen and P. H.Seeburg (1985) loc. cit.). The sequence determined using the primer isidentical to the sequence of the insert DNA of the plasmid pUC 41-2genfrom the Sau3A site in the 3' direction. The 1050 bp EcoRI DNA fragmentof the plasmid pSK⁻ 41-2gen was subcloned into the EcoRI site of thevector pKS⁺ (T. Maniatis et al., loc. cit.). A restriction map of thisDNA fragment was constructed. Based on this map, restriction fragmentswere subcloned into the bluescript vectors and, after ssDNA preparation,sequenced (Stratagene instruction manual). This entailed the sequencebeing completely determined from the EcoRI restriction site of the 41-2gene in the 3' direction as far as the Sau3A site. Table 17, which is acontinuation of Table 16, shows the DNA sequence and the derived aminoacid sequence of this EcoRI-Sau3A DNA fragment, which comprises 1050 bp,of the clone pSK⁻ 41-2gen. This gene section now codes for only 35additional amino acids until a TAG stop codon supervenes. The 3'non-coding region of the gene is extremely AT-rich (AT content=84.7%)and contains more than 11 stop codons in each of the three readingframes. The S1 mapping technique (F. M. Ausubel et al. (1987), CurrentProtocols in Molecular Biology, Harvard Medical School, Boston) revealedno evidence whatever of an intron-exon structure of this gene. Finally,Northern blot analysis (T. Maniatis et al., loc. cit.) detected an mRNAof the schizont stage with a size of 1.6 kb. It is therefore necessaryto assume that the 41-2 gene has only a single coding section of 552 bp(AT content=73%). The latter codes for an antigen of 21512 Dalton, whichhas a signal sequence (cf. Example 5) but no repetitive sections.Besides the signal sequence, this antigen has two further hydrophobicsections in amino acid positions 73 to 85 and 130 to 147, which may havea membrane-anchoring function.

Example 7

Expression of the inserts of the clones 41-1 to 41-5 and 41-7, 41-10 and41-14 in the vector pEX31

The insert fragments of the clones 41-1 to 41-5, 41-7, 41-10 and 41-14were, after restriction with EcoRI, isolated by gel electrophoresis,ligated into a vector pEX31b which had been digested with therestriction enzyme EcoRI and dephosphorylated (K. Strebel et al. (1986)Journal of Virology 57, 983-991), and transformed into competent C600bacteria containing the plasmid pCI857 (F. Remaut et al. (1981), Gene15, 81-93). Individual colonies were examined by SDS-PAGE for expressionof the Plasmodia-specific DNA sequences as MS2 polyermase fusionproteins. Induction was effected by increasing the temperature by themethod of H. K upper et al. (in Y. Ikeda and T. Beppu (ed). Proceedingsof the Fourth International Symposium on Genetics of IndustrialMicroorganisms (1982), Kyoto Kodansha Ltd., Tokyo). Expression of all 8fragments was possible in high yield.

Example 8

Purification of the expression products

Cultures of transformed bacteria were each shaken vigorously in 1 l ofLB medium containing 50 μg/ml of ampicillin and 25 μg/ml kanamycin at28° C. for 20 h. Addition of 4 l of LB medium which had been heated to42° C. was followed by renewed shaking at 42° C. for 4 h. The bacteriawere removed by centrifugation, resuspended in 200 ml ofphosphate-buffered saline (PBS) and disrupted mechanically. The solubleproteins were removed by centrifugation, and the sediments whichcontained the expression products were washed twice with PBS and thenwashed successively with increasing urea concentrations (from 1M toabout 5M) until the fusion proteins dissolved. Subsequently, dialysiswas carried out with decreasing urea concentrations until the ureaconcentration which sufficed to keep the expression products in solutionwas reached. This process resulted in a purity of 60-80%.

Example 9

Detection of the antigen encoded by 41-2gen

It was not possible, using rabbit antisera directed against theexpression product of the clone 41-2, unambiguously to detect malariaantigen in the Western blot with schizont proteins. Therefore the aimwas to express a larger DNA fragment of the gene 41-2. For this purpose,the restriction enzymes AluI and EcoRI were used to isolate a 388 bpfragment of the 41-2 gene which contains 70% of the coding region. Thisfragment was subcloned in the SmaI and EcoRI sites of the plasmid pUC18.The restriction enzyme EcoRI was used to isolate from this plasmid a 401bp fragment, which was cloned into the EcoRI site of the vector pEX31b.After transformation it was possible to identify bacterial colonieswhich express a fusion protein of 26 kD (cf. Example 7). This waspurified (cf. Example 8) and used for immunizing rabbits. The antiserarecognized, in the Western blot with schizont proteins, a 29 kD antigenwhich is encoded by the 41-2 gene. The difference between the calculatedmolecular weight of 22 kD and the molecular weight of 29 kD in SDSpolyacrylamide gels can be explained by secondary modification. Thus,the protein has an N-gly-cosylation site in the asparagine residue inposition 166.

Confirmation that the clone 41-2 encodes a very small antigen isobtained from Northern blot analysis. Thus, there was detected in theschizont stage of P. falciparum a mRNA of 1.6 kb which hybridizes withthe insert DNA of the clone 41-2. Northern blot analysis was carried outby known methods (T. Maniatis et al., loc. cit.).

Example 10

Assignment of antigens of other clones

Antisera against the purified fusion proteins of the clones 41-1, 41-3,41-4, 41-5, 41-7 and 41-10 were used to identify the correspondingantigens by Western blot analysis with schizont proteins. In this, theantisera against the fusion proteins of the clones 41-1, 41-3 and 41-4did not allow ambiguous identification. The insert DNA of the clone 41-5can be assigned to a 96 kD antigen. A group of three 96 kD antigens ofP. falciparum has been described (M. Jouin et al. (1987), Inf. Imm. 55,1387-1392). Antisera directed against an expression product of the clone41-10 recognize two antigens of 113 and 140 kD. The reaction with the113 kD antigen was identified as a cross-reaction with the SERPI antigen(cf. Example 2) which is identical to a protective antigen. 41-10 thusencodes a 140 kD protein of P. falciparum.

It is common to all these genes that the antigens, or parts thereof,encoded by them, react with a serum which is directed against a 41 kDprotein band which has a protective action. Only the clone 41-7, which,together with the clone 41-2, has the strongest reactivity with theantiserum against the 41 kD protein band, can be unambiguously assignedto a 41 kD protein.

In the Western blot with schizont proteins, antisera directed againstthe fusion protein of the clone 41-7 unambiguously recognize the 41 kDprotein band, which is also recognized by the starting serum. This clonethus appears to encode a subfragment of the 41 kD antigen. Confirmationthat the investigated clones harbor partial sequences of various geneswas moreover obtained by Southern blot analyses for the clones 41-1,41-2, 41-3, 41-7, 41-10, 41-14 and 41-15.

Example 11

Preparation of a genomic lambda gt11 gene bank

2 μg of DNA from the P. falciparum strain FCBR were incubated at 37° C.overnight with 14 units of the restriction enzyme EcoRI in 10 mMtris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol and 40% glycerol,and fractionated by gel electrophoresis. Under these conditions, therestriction enzyme shows EcoRI asterisk activity, resulting in theformation of DNA fragments from about 50 bp to 10 kb. The DNA regionbetween 500 bp and 7 kb was electroeluted and cloned by the method of T.V. Huynh et al. (1985; in DNA cloning Vol. I, a practical approach, ed.D. M. Glover) into the vector lambda gt11. A gene bank of 5×10⁵recombinant phage clones was prepared and was amplified.

Example 12

Isolation and sequencing of the 41-7 gene

Since the clone 41-7 actually codes for a subfragment of a 41 kD protein(cf. Example 10), the aim is to isolate the complete gene. For thispurpose, the genomic lambda gt11 EcoRI* gene bank (cf. Example 11) wasscreened by known methods (T. Maniatis et al., loc. cit) withnick-translated insert DNA of the clone 41-7. This resulted in threelambda gt11 clones from each of which it was possible to isolate, usingthe restriction enzymes EcoRI and SalI, and insert 3.3 kb in size. Themalaria-specific portion of the insert amounts to 2.3 kb. A restrictionmap of this DNA fragment was constructed. Based on this, subfragmentswere cloned into the bluescript vectors (Stratagene) for sequencing. Itwas possible to elucidate the complete DNA sequence of thismalaria-specific fragment which is 2.3 kb in size. Table 18 shows theDNA sequence of the 41-7 gene, with the amino acid sequence derivedtherefrom. The gene has no introns. 525 base-pairs of the 5' non-codingregion were ascertained (AT content=84.2%), as were 772 base-pairs ofthe 3' non-coding region (AT content=84.2%). The central sectioncomprises 1086 base-pairs (AT content =64.4%) and codes for 362 aminoacids. The calculated molecular weight of 39314 D for this gene agreeswell with the molecular weight of 41 kD determined by gelelectrophoresis. This gene is transcribed into an mRNA which is 2.4 kbin size, as was ascertained by Northern blot analysis by known methods(T. Maniatis et al. loc. cit.). It was possible to deduce by Southernblot analysis (cf. Example 4) that only one copy of this gene exists ineach P. falciparum genome. It was also found that this gene is conservedin various P. falciparum strains (FCBR from Colombia, Palo Alto fromUganda, SGE2 from Zaire, ItG₂ G₁ from Brazil, and VOR from Vietnam). Inaddition, the malaria-specific DNA sequence of the clone 41-7 (strainT996 from Thailand) is identical to the partial sequence from position464 to 729 of the gene isolated from the FCBR strain. Thus the clone41-7 codes for the 88 N-terminal amino acids of the 41 kD protein.

It is evident from the derived amino acid sequence that the 41 kDprotein contains no signal sequence and no repetitive sections. TheUWGCG (University of Wisconsin, Genetic Computer Group) program was usedto examine this amino acid sequence for homologous proteins within theNBRF protein data bank. It was found from this that the 41 kD protein is66% homologous with human liver aldolase (M. Mukai et al., (1985),Nucleic Acid Res. 13, 5055-5069), 66% homologous with rat liver aldolase(K. Tsutsami et al, (1984) J. Biol. Chem. 259, 14572-14575), 68%homologous with rabbit muscle aldolase (D. R. Tolan et al. (1984), J.Biol. Chem. 259, 1127-1131) and 61% homologous with the aldolase fromTrypanosoma brucei (C. E. Clayton (1985) EMBO J. 4, 2997-3003). The 41kD protein thus appears to be the P. falciparum aldolase.

Example 13

Experimental protection in an animal model: immunization of Aotuslemurinus griseimembra (karyotype VI)

This experiment was carried out to test the described expressionproducts for the efficacy in inducing protective immunity in monkeyssusceptible to P. falciparum.

The vaccine used in the experiment was a combination of the expressionproducts of the immunologically strongly reacting clones 41-1, 41-2 and41-3.

1. Design of experiment

6 Aotus monkeys of the abovementioned species (body weight 1,000-1,500g, male and female animals bred by Behringwerke AG) were randomized andassigned to 2 groups each of 3 animals.

Fusion proteins of the clones 41-1, 41-2 and 41-3 were dissolved in 3Murea in PBS and mixed in the ratio 1:1:1 (final concentration: 300 μg ofprotein/ml). 3 animals were immunized subcutaneously 3×, at intervals of3 weeks, each time with 1 ml of the combined fusion proteins. A 10%admixture of 50% Al(OH)₃ /45% lecithin/5% saponin to the antigen wasused as adjuvant.

3 animals in the infection control group likewise each received aninjection of 3M urea in PBS+adjuvant without antigenic component inaccordance with the abovementioned scheme.

In order to ensure that the experimental P. falciparum infections of theanimals were as near the same severity as possible, all the monkeys weresplenectomized eight days after the last immunization (increasedsusceptibility).

On day 67 after the 1st vaccination, all 6 animals were infectedintravenously with 5×10⁶ parasitized erythrocytes. The strain chosen forchallenge was P. falciparum Palo Alto (Uganda) which as adapted in vitroto Aotus erythrocytes and was transferred directly from a splenectomizeddonor animal (4% parasitemia) to the experimental animals. This strainis distinguished by high infectiosity compared with other P. falciparumisolates. It is also of interest to mention that this strain isheterologous in terms of provenance and serotype to the strain T996(Thailand) used for obtaining the immunization antigens.

Physiological, parasitological, serological and clinicochemicalparameters were examined regularly during the entire course of the study(before and after immunization and after the challenge).

2. Results

No phathological changes in any of the physiological (clinicalmanifestations, temperature, weight) or clinicochemical (erythrocytes,hematocrit, ESR, serum enzymes GPT and GOT) parameters investigated wereseen throughout the immunization period. Additional drug-safetyinvestigations (acute subcutaneous toxicity in the mouse, localtolerability in the monkey in accordance with the specifications of theEuropean Pharmacopoeia) demonstrated adequate safety and tolerability ofthe vaccine preparation used.

2.1 Parasitemia

The main parameter for assessing the value of an induced protection isthe detection under the microscope of parasitized erythrocytes in theperipheral blood of the experimental animal.

A few parasitized erythrocytes (less than 1 per thousand) weredetectable in the Giemsa-stained blood smear from the non-immunizedanimals as early as 7-10 days after the infection. The appearance ofparasites in the immunized animals was delayed to 10-15 days afterinfection, and they reached maximum parasitemias of 1-2% and controlledthe infection spontaneously. There was one intercurrent death of ananimal from pneumonia.

Whereas one animal in the non-immunized group was able itself to controla maximum parasitemia of 4.5%, it was necessary for the two otheranimals to be treated with mefloquine (Hoffman La Roche) after aparasitemia of >10% had been reached, in order to prevent the infectiontaking a lethal course. The Palo Alto strain used for the challengeproved in preceding infection experiments to be chloroquine-resistant.

FIG. 1A, on the left, shows the course of the parasitemia in Aotusmonkeys after immunization with a combination vaccine composed of fusionproteins of the clones 41-1, 41-2 and 41-3 and, FIG. 1B on the right,shows that of the control (non-immunized animals).

                                      TABLE 1                                     __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-1, and derived amino acid sequence                               __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                     __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-2, and derived animo acid sequence                               __________________________________________________________________________     ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                    __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-3, and derived amino acid sequence                               __________________________________________________________________________     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                    __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-4, and derived amino acid sequence                               __________________________________________________________________________     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                    __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-5, and derived amino acid sequence                               __________________________________________________________________________     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                    __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-6, and derived amino acid sequence                               __________________________________________________________________________     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                    __________________________________________________________________________

                                      TABLE 7                                     __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-7, and derived amino acid sequence                               __________________________________________________________________________     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                    __________________________________________________________________________

                                      TABLE 8                                     __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-8, and derived amino acid sequence                               __________________________________________________________________________     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                    __________________________________________________________________________

                                      TABLE 9                                     __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    malaria-specific insert DNA of the clone 41-9                                 __________________________________________________________________________     ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                     ##STR40##                                                                    __________________________________________________________________________

                                      TABLE 10                                    __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    malaria-specific insert DNA of the clone 41-10                                __________________________________________________________________________     ##STR41##                                                                     ##STR42##                                                                     ##STR43##                                                                    __________________________________________________________________________

                                      TABLE 11                                    __________________________________________________________________________    Nucleotide sequence of the malaria-specific DNA insert of                     the clone 41-12, and derived animo acid sequence                              __________________________________________________________________________     ##STR44##                                                                     ##STR45##                                                                     ##STR46##                                                                    __________________________________________________________________________

                                      TABLE 12                                    __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    malaria-specific insert of clone 41-13.                                       __________________________________________________________________________     ##STR47##                                                                     ##STR48##                                                                     ##STR49##                                                                     ##STR50##                                                                     ##STR51##                                                                     ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                     ##STR55##                                                                     ##STR56##                                                                     ##STR57##                                                                    __________________________________________________________________________

                                      TABLE 13                                    __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    malaria-specific insert of clone 41-14.                                       __________________________________________________________________________     ##STR58##                                                                     ##STR59##                                                                     ##STR60##                                                                     ##STR61##                                                                     ##STR62##                                                                     ##STR63##                                                                     ##STR64##                                                                     ##STR65##                                                                     ##STR66##                                                                    __________________________________________________________________________

                                      TABLE 14                                    __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    malaria-specific insert of clone 41-15.                                       __________________________________________________________________________     ##STR67##                                                                     ##STR68##                                                                     ##STR69##                                                                     ##STR70##                                                                     ##STR71##                                                                    __________________________________________________________________________

                                      TABLE 15                                    __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    malaria-specific insert of clone 41-17                                        __________________________________________________________________________     ##STR72##                                                                     ##STR73##                                                                    __________________________________________________________________________

                                      TABLE 16                                    __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    5' end of the P. falciparum (isolate FCBR) gene specified                     by the insert of the clone 41-2.                                              __________________________________________________________________________     ##STR74##                                                                     ##STR75##                                                                     ##STR76##                                                                     ##STR77##                                                                     ##STR78##                                                                     ##STR79##                                                                     ##STR80##                                                                     ##STR81##                                                                     ##STR82##                                                                     ##STR83##                                                                     ##STR84##                                                                     ##STR85##                                                                     ##STR86##                                                                     ##STR87##                                                                     ##STR88##                                                                     ##STR89##                                                                     ##STR90##                                                                     ##STR91##                                                                     ##STR92##                                                                     ##STR93##                                                                     ##STR94##                                                                    __________________________________________________________________________

                                      TABLE 17                                    __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    3' end of the P. falciparum (isolate FCBR) gene specified                     by the insert of the clone 41-2.                                              __________________________________________________________________________     ##STR95##                                                                     ##STR96##                                                                     ##STR97##                                                                     ##STR98##                                                                     ##STR99##                                                                     ##STR100##                                                                    ##STR101##                                                                    ##STR102##                                                                    ##STR103##                                                                    ##STR104##                                                                    ##STR105##                                                                    ##STR106##                                                                    ##STR107##                                                                    ##STR108##                                                                    ##STR109##                                                                    ##STR110##                                                                    ##STR111##                                                                    ##STR112##                                                                   __________________________________________________________________________

                                      TABLE 18                                    __________________________________________________________________________    Nucleotide sequence and derived amino acid sequence of the                    41 kD protein of P. falciparum                                                __________________________________________________________________________     ##STR113##                                                                    ##STR114##                                                                    ##STR115##                                                                    ##STR116##                                                                    ##STR117##                                                                    ##STR118##                                                                    ##STR119##                                                                    ##STR120##                                                                    ##STR121##                                                                    ##STR122##                                                                    ##STR123##                                                                    ##STR124##                                                                    ##STR125##                                                                    ##STR126##                                                                    ##STR127##                                                                    ##STR128##                                                                    ##STR129##                                                                    ##STR130##                                                                    ##STR131##                                                                    ##STR132##                                                                    ##STR133##                                                                    ##STR134##                                                                    ##STR135##                                                                    ##STR136##                                                                    ##STR137##                                                                    ##STR138##                                                                    ##STR139##                                                                    ##STR140##                                                                    ##STR141##                                                                    ##STR142##                                                                    ##STR143##                                                                    ##STR144##                                                                    ##STR145##                                                                    ##STR146##                                                                    ##STR147##                                                                    ##STR148##                                                                    ##STR149##                                                                    ##STR150##                                                                    ##STR151##                                                                    ##STR152##                                                                   __________________________________________________________________________

We claim:
 1. A purified and isolated DNA sequence encoding an amino acidsequence as shown in Table 3 or a fragment of said DNA sequence encodinga polypeptide reactive with antiserum to a 41 kDa Plasmodium falciparumband.
 2. A purified and isolated DNA sequence encoding an amino acidsequence as shown in Table 2 or a fragment of said DNA sequence encodinga polypeptide reactive with antiserum to a 41 kDa Plasmodium falciparumband.
 3. A vector comprising a DNA sequence as claimed in claim
 1. 4. Avector comprising a DNA sequence as claimed in claim
 2. 5. A host cellcomprising a DNA sequence as claimed in claim
 1. 6. A host cellcomprising a DNA sequence as claimed in claim
 2. 7. A process for thepreparation of a purified and isolated polypeptide that reacts withantiserum to a 41 kDa Plasmodium falciparum band comprising expressionof the DNA sequence of claim
 1. 8. A process for the preparation of apurified and isolated polypeptide that reacts with antiserum to a 41 kDaPlasmodium falciparum band comprising expression of the DNA sequence ofclaim 2.